NPM1基因沉默的HL-60及其耐药细胞株的建立

Establishment of Nucleophosmin Gene Silenced HL-60 and Its Resistant Cell Line

本研究旨在构建针对HL-60细胞及其耐药细胞株(HL-60/ADR)的NPM1-RNAi的细胞模型,为研究NPM1基因在白血病耐药过程中的作用奠定实验基础。通过针对NPM1基因的shRNA与线性化的pGCSIL-GFP载体进行连接转化,对获得的重组子(pGCSIL-GFP-NPM1-shRNA)进行PCR和测序鉴定。采用慢病毒载体系统将pHelper 1.0载体、pHelper 2.0载体与pGCSIL-GFP-NPM1-shRNA共转染293T细胞,包装成NPM1-RNAi-LV,将慢病毒载体感染HL-60及HL-60/ADR细胞。采用实时定量RT-PCR和Western blot法分别从mRNA和蛋白水平验证建立的细胞株的转染效率。结果表明,成功构建了重组真核表达载体pGCSIL-GFP-NPM1-shRNA,并通过慢病毒载体系统包装成NPM-1-RNAi-LV;将NPM1-RNAi-LV转染至HL-60及HL-60/ADR细胞后,从mRNA水平上看,对细胞的NPM1 mRNA表达有显著抑制,达到90%以上(p<0.05);从蛋白水平上看,对细胞的NPM蛋白表达具有显著的抑制效果,表明转染后的HL-60及HL-60/ADR细胞的NPM1基因特异性沉默。NPM1基因沉默后,HL-60/ADR对阿霉素的耐药性有一定程度的下降。结论:成功构建了NPM1-RNAi的HL-60及HL-60/ADR细胞模型,为进一步研究NPM1基因在白血病耐药过程中的作用建立了良好的实验基础。

This study was aimed to construct model cell line of NPM1-RNAi in HL-60 cells and its resistant line（HL-60/ADR） so as to provide a experimental basis for investigating the potential role of NPM1 gene in leukemia drug resistance.The shRNA targeting to NPM1 was ligated into linear pGCSIL-GFP vector,and transformed into E.coli DH5α.Positive clone was identified by PCR and DNA sequencing.pHelper 1.0,pHelper 2.0 and pGCSIL-GFP-NPM1-shRNA were cotransformed into 293T cells by lentivius vector system.NPM1-RNAi-LV was transfected into HL-60 and HL-60/ADR cell lines.The efficiency of NPM-RNAi-LV was detected by using real-time quantitative RT-PCR and Western blot.The results showed that the recombinant eukaryotic expression vector pGCSIL-GFP-NPM1-shRNA was constructed.pGCSIL-GFP-NPM1-shRNA was packed into NPM1-RNAi-LV by lentivirus vector system,and transfected into HL-60 and HL-60/ADR cell lines.At mRNA level,the efficiency of NPM1 mRNA knockdown was more than 90%（p〈0.05）.At protein level,obvious down-regulation of NPM protein was noted,indicating that NPM1 gene in HL-60 and HL-60/ADR cell lines was knocked down after transfected with NPM1-RNAi-LV.The resistance of HL-60/ADR cell line to adriamycin decreased to a certain degree after NPM1 gene silencing.It is concluded that the model cell lines of NPM1-RNAi in HL-60 and HL-60/ADR are successfully constructed,which can be used for investigating the potential role of NPM1 gene in drug resistance of leukemia.